PROJECT SUMMARY/ABSTRACT Metalloenzymes orchestrate complex multiproton/multielectron reactions critical to human life, such as the 6H+/6e- reduction of dinitrogen to ammonia by nitrogenase and the 4H+/4e- reduction of dioxygen to water by monooxygenase enzymes. To accomplish these reactions with minimal loss of partially reduced species (PRS), many metalloenzymes have redox-active cofactors located in close proximity to the active site. These cofactors are often loaded with several electron and proton equivalents prior to substrate binding, enabling selective conversion of the substrate to product with minimal PRS loss. Often, PRSs are highly reactive and lead to cellular damage. For enzymes such as Cytochrome P450 (CYP) and Cytochrome c Oxidase (CcO), loss of PRSs, such as H2O2, is linked to various diseases such as down syndrome, multiple sclerosis and cancer. Therefore, understanding the influence that local electron reservoirs have on the selectivity of multiproton/multielectron transformations may aid the treatment of the aforementioned diseases. In order to fundamentally understand the influence that local electron reservoirs have on the selectivity of multielectron/multiproton transformations, we propose to study the activity and selectivity for O2 reduction using iron porphyrins covalently attached to conductive electrodes as artificial models of O2 reducing enzymes. Rather than using a molecular electron reservoir, we will covalently attach metalloporphyrins to carbon and metal oxide electrode surfaces. We hypothesize that metalloenzymes utilize these redox-loaded cofactors to provide the active site with a highly coupled source of electrons, and that changes to the electron coupling between the donor (electrode) and acceptor (metalloporphyrin) will influence the kinetics of the bifurcating steps leading to the desired product (H2O) or the undesired PRS (H2O2). By using an electrode as a tunable surrogate for a redox cofactor, these interfacial constructs will allow a multidimensional control of the distance, coupling and electron transfer driving force between the electrode and iron porphyrin active site, enabling a fundamental study of the steps that lead to bifurcation and loss of PRS in enzymes such as CcO and CYP. These studies will provide insights into how redox-active cofactors influence product bifurcation in metalloenzymes, which may lead to new methods of treatment or prevention of diseases induced by H2O2 loss in biological systems.